The Analysis of MDMA through Impurity Profiling and Hair and Urine Sampling
Introduction
The origins of 3,4-methylenedioxymethamphetamine (MDMA), or ecstasy, a ring-substituted amphetamine derivative are not completely known. [1] The first documented synthesis of MDMA was by the German pharmaceutical company Merck in 1912 as an appetite suppressor.[1] Classified as a schedule 1 hallucinogen by the U.S. government, MDMA has increased in popularity as a recreational drug during the past twenty years.[2] Also known as Adam, baby slits, chocolate chips, clarity, doctor, essence, kleenex, and roll, MDMA is an enactogen. [2] An enactogen has some similiarities between hallocinogens and stimulants except that it provides an emotional and social closeness to individuals that are lacked in other drugs. [3] Extended use of MDMA has been associated with changes in makers in neurotransmitter systems of individuals, specifically serotonin 5-HT receptors. [4] MDMA increases release and inhibits reuptake of serotonin to cause the associated effects of euphoria, empathy, sociability, and mild hallucinations. [5] In order to identify the specific strain and amount of MDMA ingested by an individual, the pathways used in synthesis along with the chemical analysis of sample hair and urine must be understood.
MDMA Synthetic Pathways
In clandestine laboratories, the most popular method of MDMA synthesis is the reductive amination of of 3,4-methylenedioxyphenylpropan-2-one, or MDP-2-P, because of the various reducing agents possible for use. [6] The three most common reductive amination reactions are borohydride reduction at low temperatures, dissolving a metal reduction in an aluminum mercury amalgam, and cyanoborohydride reduction (Fig. 1). [7] The two other synthesis reactions are the Leukart reaction (Fig. 2) and the safrole bromination reaction (Fig. 3). [7]
Figure 1. The reductive amination of MDP-2-P through three separate reagents to form MDMA. [7]
Figure 2. The Leukart reaction of MDP-2-P to produce MDMA.
Use of Impurities to Determine the Synthetic Pathway
In order to understand what synthetic pathway was used to synthesize the MDMA, the impurities associated with the sample must be identified. [11] The final synthesized product depends on the manufacturing process which includes the synthesis pathway, the reagents, and the quality of the precursors (most commonly MDP-2-P). [8] Impurity profiling has two main purposes: to determine which synthesis route was taken and to compare seized samples of MDMA. [9] These impurities serve as synthesis markers as certain synthesis routes have particular impurities. [9] However, an ideal marker of where the impurity exists only for a specific pathway is highly unlikely. For example, N-formylMDMA is a known marker for the Leukart reaction, the safrole bromination reaction, and the reductive amination reactions. [9] Therefore, the more synthetic markers in a sample, the easier it will be to identify which synthetic pathway was taken to create the sample. Profiling sheds information about various reagents and catalysts used in production, along with various methods of purification of the crude product. [10] There are limitations to this approach in real world applications. For instance, if a sample of MDMA was purified, some of the characteristic impurities would be removed, thus making it difficult to specify which synthetic pathway was taken. [9]
Because MDP-2-P is challenging to obtain in the real world, it is easier to synthesized the precursor when in a clandestine laboratory [11]. The most common methods include the oxidation of isosafrole in an acid medium and the condensation of piperonal and nitroethane to yield 1-(3,4-methylenedioxyphenyl)-2-nitropropene which can be reduced (Fig. 4). [9]
Figure 4. The two most common synthesis reactions of MDP-2-P, a common precursor to MDMA. [9]
The recent crackdowns on illicit precursors have led to creative methods to reach the final product. Two popular solutions have arisen as precursors: piperine, a component of pepper, and vanillin, a common perfume and flavoring additive. [11] From these two materials, piperonal can be prepared (Fig. 5). Piperine can produce piperonal via two routes: oxidative cleavage with aqueous KMnO4 in THF or oxidative cleavage by ozonolysis. [11] In oxidative cleavage with KMnO4, KMnO4 is added dropwise to a solution of piperine/THF. After about four hours of mixing, MnO2 precipitates out, leaving a yellow solution of which the sample can be extracted with diethyl ether. The entire process took approximately ten hours according to Gallagher et al. Ozonolysis requires a stream of ozone gas to pass through a solution of piperine, water, and acetone, for about eight hours. Afterwards, the sample was extracted with diethyl ether, dried, and evaporated to produce bright yellow oil. Ozonolysis produced the greater yield in less time, with fewer impurities [11].
To produce piperonal from vanillin, a mixture of vanillin and toluene was combined with aluminum chloride. [11] Pyridine was added dropwise at reflux for about six hours. Dilute HCl and diethyl ether were used to extract the sample of 3,4-dihydroxybenzaldehyde from vanillin. A solution of 3,4-dihydroxybenzaldehyde and dichloromethane was added to a solution of potassium carbonate and N-methyl-pyrrolidione, and stirred for three hours over heat. After extraction with toluene and vacuum evaporation, the yield was greater than that of cleavage of piperine with aqueous KMnO4/THF, but less than that of ozonolysis. [11] The steps to obtain MDP-2-P from piperonal resemble those of Figure 4.
Figure 5. Preparation of piperonal from piperine and vanillin, respectively.
Based on the precursors, intermediates, and by-products of the sample MDMA, the synthetic pathway can be determined. These impurities, as previously stated, are synthetic markers and are present in reaction mixtures. The presence of certain impurities, such as safrole and isosafrole, suggest that oxidation of safrole or isosafrole in an acid medium was used to produce MDP-2-P [9]. If 1,2-(methylenedioxy)phenylpropan-2-ol is found, the compound was most probably oxidized from safrole or isosafrole as this is the hydrogenated form of MDP-2-P. [6] Some of the more common synthetic markers and their associated pathway are depicted in Figure 6. There is overlap between which impurities identify which pathway. The green box shows the overlapping impurities associated with the Leukart reaction and the safrole bromination reaction. The red box represents the common impurities of reductive amination and the Leukart reaction. Although these are the more common impurities, the increased restrictions on available chemicals result in new means of producing MDMA, and thus varying impurities. As such, more research would be necessary to be able to classify the seized sample synthesis pathway.
Figure 6. Common impurities associated with their synthetic pathway.
Various Analytical Techniques Associated with Impurity Profiling
Impurity profiling can be done using a variety of analytical techniques, but the most common one is the Gas Chromatography/Mass Spectrometry method. Gas chromatography has two phases: a mobile phase and a stationary phase. [12] The stationary phase is a column made of metal which contains a microscopic layer of liquid or a packed solid. [13] The mobile phase, usually an inert gas stream, will move the sample through the column where the components would be separated by heat [12]. By heating the sample, components will vaporize to pass through the stationary phase to be detected by a detector. [12] Gas chromatography is coupled with mass spectrometry because as GC separates the compounds in the sample, MS identifies the compounds based on their fragmentation pattern. [14] The fragmentation pattern is a result of a stream of electrons hitting the compound causing them to break off into fragments. [14] The resulting data shows the signal areas of the impurity to give a raw profile of the compound. [8] In other words, the composition of the impurity can be determined using the GC-MS method. Therefore, the simultaneous identification of a group of synthetic markers will determine the synthetic pathway used in the manufacturing of the sample. For example, if the following impurities were present: N-cyclohexyl-acetamide, N,N-Dimethylpiperonylamine, 3,4-Methylenedioxyamphetamine, N-Methylpiperonylamine, 2-Methyl-(6,7-methylenedioxyphenyl)-3-methylmorpholine, 3-Methyl-6,7-methylenedioxyisoquinoline-1,4-dione, the sample was synthesized from MDP-2-P by reduction of 1-(3,4-methylenedioxyphenyl)-2-nitropropene from piperonal, and the MDMA was synthesized by reductive amination. [9] Since N-cyclohexylacetamide was the product of a catalyst and acetic acid, the compound can be any N-alkylacetamide corresponding to the amine used as the catalyst; thus the synthetic pathway would still be reductive amination of MDP-2-P from piperonal. [9]
Palhol et al. analyzed 29 extracted impurities from 52 seized tablets using methylene chloride under basic conditions. [15] The extracts were then analyzed by GC with flame ionization detection to identify the sample’s precursors, intermediates, and by-products. [15] The components separated from the GC pass through a hydrogen flame to ionize the gas in order to determine its concentration. [16] The results of GC-FID were confirmed through GC-MS. The precursor was found to be MDP-2-P as it was found in over three fourths of the tablets. The intermediates and by-products suggested that most of the MDMA was synthesized via reductive amination, while the rest were done through Leukart synthesis. The similar impurities suggested that the tablets were manufactured by the same laboratory. It was found to be similar to seized batches from Northern France in previous years. [15]
Kochana et al. determined that the use of Solid Phase Extraction-Thin Layer Chromatography (SPE-TLC) gave the best results when profiling 1-(3,4-methylenedioxyphenyl)-2-nitropropene from piperonal. [17] This compound can be reduced to give MDP-2-P, the precursor for MDMA in both Leukart and reductive animation synthesis. The impurity was extracted in C18 columns, and TLC separation occurred on silica gel 60 plates with fluorescent indicator F254. It was concluded that a concentrated extraction provided better profiling, and that a 2:8 acetonitrile-chloroform mixture was the best possible mobile phase for TLC separation. [17] However, the limitations of this analytical technique reduce the possibility of the its use in the real world as it only applies to the 1-(3,4-methylenedioxyphenyl)-2-nitropropene intermediate.
Carter et al. had used GC-Isotope Ratio Mass Spectrometry to analyze the δ13C values for extracted MDMA from four tablets. [18] The MDMA was then oxidized to MDP-2-P and piperonal of which the δ13C values were taken. Since the δ13C value was more concentrated for piperonal than MDP-2-P, it was suggested that the MDMA tablets were synthesized from piperonal and not from the oxidation of isosafrole. [18] A follow up study by Carter et al. determined the δ13C, δ2H, and δ15N values of MDMA from five different seized tablets. [19] Based on the results, when considering the δ13C values, three batches were isotopically distinct. Four batches were distinct based on δ2H. [19] However, each batch was completely distinct when all three values were taken into account. This suggests that the isotropic analysis of active ingredients in MDMA can be used as a “fingerprint” of the sample, which could be connected back to the original batch.
The four analytical techniques discussed above were GC-MS, GC-MS with flame ionization detection, SPE-TLC, and GC-ISRMS. Each of these techniques has their own advantages and disadvantages. The problem with GC-MS is that it does not work for non volatile components. However, it has been the most used method in impurity profiling so it does have a definitive procedure and protocol. Also, it provides an explicit analysis of what compounds are present in the sample. The use of flame ionization detection along with GC-MS allows for greater sensitivity towards hydrocarbons. [20] Flame ionization allows organic compounds with greater carbon atoms than nitrogen, phosphorus, or sulfur atoms to be detected better because of the sensitivity of FID to high carbon concentrations. [16] The disadvantage of FID would be that the sample would be destroyed. [16] However, in a situation where a large batch of MDMA would be seized, this would not be a problem. Moreover, the sensitivity of FID depends on certain aspects, such as the rate flow of the carrier gas, the rate flow of the combustion gas, the diameter of the jet the flame is produced by, and the position of the jet to the detector. [16] As such, if the compound is not a hydrocarbon, like a highly oxygenated compound, the sensitivity of FID decreases because the combustibility of the compound is lower. [20] The SPE-TLC technique was great when profiling a specific precursor, 1-(3,4-methylenedioxyphenyl)-2-nitropropene from piperonal, however, it would need to be modified for each impurity. So as an impurity profiling technique, it falls short in terms of practicality. GC-ISRMS provided the greatest amount of distinction between batches because it defined each batch according to three isotopic values, δ13C, δ2H, and δ15N. Although, it provided the best “fingerprint” of the MDMA samples seized, it was performed under the assumption that MDMA was oxidized from MDP-2-P and piperonal. After reviewing each of the techniques, GC-MS would be the best technique to identify the impurities associated with whichever synthetic pathways. Because the literature on GC-MS is already prevalent and it provides definitive results of the components in the sample, it would be a good identifier of impurities in a sample. From these impurities, the synthetic pathway can be deduced easier. But in order to identify if a sample was from a specific batch and the synthetic pathway has already been deduced, GC-IRMS would be best. Although superfluous, it would provide a strong support of the GC-MS results. Even though FID has the greatest sensitivity to identify impurities, it should be used as a last resort as this technique destroys the sample.
Cheng et al. used a combination of infrared spectroscopy, high performance liquid chromatography, liquid chromatrgraphy electrospray ionization mass spectrometry, and GC-MS to identify the impurities in 600,000 tablets seized in Hong Kong. [21] Their analysis found the most common impurities to be N-formyl MDMA, 3,4-methylenedioxyN-methylbenzylamine, 3,4-methylenedioxyphenyl-2-propanol, and MDP-2-P. From their results, reductive amination using cyanoborohydrate and the Leukart synthetic pathways were found to be most prevalent in the samples. [21] The use of multiple techniques to identify synthetic pathways of numerous samples seems to be most efficient because the limitations of one technique can be overcome through the use of another.
How to Identify the Presence of MDMA in an Individual
The identification of impurities of MDMA helped to determine which synthetic pathway was used to produce the tablet. But how would you identify if MDMA was in an individual’s system? Various analytical methods have been applied to samples of hair and urine to determine the presence of MDMA. Urine and hair samples are used mainly because they lack the complexities of blood or organ samples. [22] When comparing hair relative to urine samples, hair samples have a greater percentage of being positive to MDMA than urine. [23] In other words, MDMA will test positive in urine for frequent users, but will not test positive for occasional users. However, if hair is analyzed, occasional users would test positive for the compound. Opiates and amphetamines have a relatively short window of indication in urine, about two to three days; whereas hair can indicate upwards of three months, possibly longer. [24] The use of hair or urine sample depends on what is being analyzed. For instance, Lee et al. used hair samples to identify chronic use of MDMA in anorectics. [25] As such, the month by month use and amount of MDMA can be identified in hair samples since it grows at a rate of about one centimeter per month. [25] Although a urine sample would also determine the presence of MDMA in the system (if the anorectic was a frequent user), it would not provide the same information as a hair sample. A sample of urine or hair is tested for not only MDMA but their metabolites as well, such as 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-dihydroxymethamphetamine, 3-hydroxy-4-methoxymethamphetamine. [26] HMMA is the product of o-methylation of 3,4-dihydroxymethamphetamine and is considered the main metabolite of interest in urine. [26] However, since HMMA is easily hydrolyzed, a majority of the compound is excreted as glucuronide or sulfate so it is difficult to analyze for HMMA. [27]
The analytical technique usually associated with urine and hair analysis is GC-MS. [22] In order for the samples of hair and urine to be analyzed, they must be prepared for GC-MS. [23] The method utilized by Han et al. on the urine samples was to use fluorescence polarization immunoassays followed by GC-MS analysis. [23] The hair was first washed twice with water than twice by methanol, incubated in methanol with 1% hydrochloric acid, evaporated, incubated with ethyl acetate and TFAA, cooled, evaporated, and then mixed with methanol. [23] GC-MS was used to analyze the hair samples of 791 subjects that were suspected of using MDMA. [23] The results from GC-MS analysis identified 44 individuals with MDMA or MDA in their hair, and from that, 9 were identified with MDMA or MDA in their urine. [23] Although a successful procedure, high performance liquid chromatography with a fluorescence detector could be used in place of GC-MS when analyzing urine. [28] MDMA, along with many other methylenedioxylated amphetamines, fluoresces. [22] As such, a fluorescence detector would have high selectivity towards such fluorescent compounds. Since these compounds naturally fluoresce, the derivation process in GC-MS would not be required. [28] This would decrease the cost of analyzing urine, making it more accessible to companies and countries with limited funds. [22] Another cost saving method would be gas chromatography coupled with flame ionization detection. [29] Although not as sensitive as GC-MS, GC-FID is sensitive enough to detect MDMA, MDA, and MA in urine samples. [29] It reduces the cost of identifying the compound as most laboratories have the equipment to carry out the procedure. Furthermore, by coupling GC-FID with solid phase extraction, the concentration of MDMA, MDA, and MA can be determined from urine samples. [30] Based on the components found in the samples, the purity of the tablets can also be determined. [29] For instance, if only MDMA was found after SPE and GC-FID, the tablet consumed was purely MDMA if time of collection of the sample was taken into account. [30] In other words, if the sample collected took some time to be analyzed, some of the other compounds, like MDA, could have been reduced to levels undetectable by GC-FID. For hair analysis specifically, liquid chromatography-mass spectrometry followed by mass spectrometry is becoming more popular than GC-MS because of its increased sensitivity along with lack of product loss during the derivatization step. [25] Due to the volatility associated with amphetamines, during the derivatizaiton step, evaporation occurs, leading to the loss of MDMA and inaccurate results. [25]
Conclusion
MDMA, a classified illegal enactogen, has become a prevalent recreational drug as it induces increased serotonin release while hindering synaptic reuptake. [5] The surge in popularity of the drug is accompanied by its production in clandestine laboratories. As such, methods to determine the synthesis of MDMA by analyzing the impurities associated with a sample has linked certain batches of seized MDMA to certain labs [8-11]. Various methods of analyzing the impurities include GC-MS, GC-MS with FID, SPE-TLC, and GC-IRMS. By considering a group of impurities together, the synthetic pathway can be determined [9]. Although much has been accomplished MDMA analysis, further research is needed to determine more impurities associated with various creative synthetic pathways. Not only can MDMA be analyzed in impurity profiling, but the presence of MDMA can be analyzed in human urine and hair. While GC-MS is the prevalent method, several other techniques have become increasingly popular such as GC-FID and LC-MS/MS as new approaches are being explored. [25]
References
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The Analysis of MDMA through Impurity Profiling and Hair and Urine Sampling
Introduction
The origins of 3,4-methylenedioxymethamphetamine (MDMA), or ecstasy, a ring-substituted amphetamine derivative are not completely known. [1] The first documented synthesis of MDMA was by the German pharmaceutical company Merck in 1912 as an appetite suppressor.[1] Classified as a schedule 1 hallucinogen by the U.S. government, MDMA has increased in popularity as a recreational drug during the past twenty years.[2] Also known as Adam, baby slits, chocolate chips, clarity, doctor, essence, kleenex, and roll, MDMA is an enactogen. [2] An enactogen has some similiarities between hallocinogens and stimulants except that it provides an emotional and social closeness to individuals that are lacked in other drugs. [3] Extended use of MDMA has been associated with changes in makers in neurotransmitter systems of individuals, specifically serotonin 5-HT receptors. [4] MDMA increases release and inhibits reuptake of serotonin to cause the associated effects of euphoria, empathy, sociability, and mild hallucinations. [5] In order to identify the specific strain and amount of MDMA ingested by an individual, the pathways used in synthesis along with the chemical analysis of sample hair and urine must be understood.MDMA Synthetic Pathways
In clandestine laboratories, the most popular method of MDMA synthesis is the reductive amination of of 3,4-methylenedioxyphenylpropan-2-one, or MDP-2-P, because of the various reducing agents possible for use. [6] The three most common reductive amination reactions are borohydride reduction at low temperatures, dissolving a metal reduction in an aluminum mercury amalgam, and cyanoborohydride reduction (Fig. 1). [7] The two other synthesis reactions are the Leukart reaction (Fig. 2) and the safrole bromination reaction (Fig. 3). [7]Use of Impurities to Determine the Synthetic Pathway
In order to understand what synthetic pathway was used to synthesize the MDMA, the impurities associated with the sample must be identified. [11] The final synthesized product depends on the manufacturing process which includes the synthesis pathway, the reagents, and the quality of the precursors (most commonly MDP-2-P). [8] Impurity profiling has two main purposes: to determine which synthesis route was taken and to compare seized samples of MDMA. [9] These impurities serve as synthesis markers as certain synthesis routes have particular impurities. [9] However, an ideal marker of where the impurity exists only for a specific pathway is highly unlikely. For example, N-formylMDMA is a known marker for the Leukart reaction, the safrole bromination reaction, and the reductive amination reactions. [9] Therefore, the more synthetic markers in a sample, the easier it will be to identify which synthetic pathway was taken to create the sample. Profiling sheds information about various reagents and catalysts used in production, along with various methods of purification of the crude product. [10] There are limitations to this approach in real world applications. For instance, if a sample of MDMA was purified, some of the characteristic impurities would be removed, thus making it difficult to specify which synthetic pathway was taken. [9]Because MDP-2-P is challenging to obtain in the real world, it is easier to synthesized the precursor when in a clandestine laboratory [11]. The most common methods include the oxidation of isosafrole in an acid medium and the condensation of piperonal and nitroethane to yield 1-(3,4-methylenedioxyphenyl)-2-nitropropene which can be reduced (Fig. 4). [9]
The recent crackdowns on illicit precursors have led to creative methods to reach the final product. Two popular solutions have arisen as precursors: piperine, a component of pepper, and vanillin, a common perfume and flavoring additive. [11] From these two materials, piperonal can be prepared (Fig. 5). Piperine can produce piperonal via two routes: oxidative cleavage with aqueous KMnO4 in THF or oxidative cleavage by ozonolysis. [11] In oxidative cleavage with KMnO4, KMnO4 is added dropwise to a solution of piperine/THF. After about four hours of mixing, MnO2 precipitates out, leaving a yellow solution of which the sample can be extracted with diethyl ether. The entire process took approximately ten hours according to Gallagher et al. Ozonolysis requires a stream of ozone gas to pass through a solution of piperine, water, and acetone, for about eight hours. Afterwards, the sample was extracted with diethyl ether, dried, and evaporated to produce bright yellow oil. Ozonolysis produced the greater yield in less time, with fewer impurities [11].
To produce piperonal from vanillin, a mixture of vanillin and toluene was combined with aluminum chloride. [11] Pyridine was added dropwise at reflux for about six hours. Dilute HCl and diethyl ether were used to extract the sample of 3,4-dihydroxybenzaldehyde from vanillin. A solution of 3,4-dihydroxybenzaldehyde and dichloromethane was added to a solution of potassium carbonate and N-methyl-pyrrolidione, and stirred for three hours over heat. After extraction with toluene and vacuum evaporation, the yield was greater than that of cleavage of piperine with aqueous KMnO4/THF, but less than that of ozonolysis. [11] The steps to obtain MDP-2-P from piperonal resemble those of Figure 4.
Based on the precursors, intermediates, and by-products of the sample MDMA, the synthetic pathway can be determined. These impurities, as previously stated, are synthetic markers and are present in reaction mixtures. The presence of certain impurities, such as safrole and isosafrole, suggest that oxidation of safrole or isosafrole in an acid medium was used to produce MDP-2-P [9]. If 1,2-(methylenedioxy)phenylpropan-2-ol is found, the compound was most probably oxidized from safrole or isosafrole as this is the hydrogenated form of MDP-2-P. [6] Some of the more common synthetic markers and their associated pathway are depicted in Figure 6. There is overlap between which impurities identify which pathway. The green box shows the overlapping impurities associated with the Leukart reaction and the safrole bromination reaction. The red box represents the common impurities of reductive amination and the Leukart reaction. Although these are the more common impurities, the increased restrictions on available chemicals result in new means of producing MDMA, and thus varying impurities. As such, more research would be necessary to be able to classify the seized sample synthesis pathway.
Various Analytical Techniques Associated with Impurity Profiling
Impurity profiling can be done using a variety of analytical techniques, but the most common one is the Gas Chromatography/Mass Spectrometry method. Gas chromatography has two phases: a mobile phase and a stationary phase. [12] The stationary phase is a column made of metal which contains a microscopic layer of liquid or a packed solid. [13] The mobile phase, usually an inert gas stream, will move the sample through the column where the components would be separated by heat [12]. By heating the sample, components will vaporize to pass through the stationary phase to be detected by a detector. [12] Gas chromatography is coupled with mass spectrometry because as GC separates the compounds in the sample, MS identifies the compounds based on their fragmentation pattern. [14] The fragmentation pattern is a result of a stream of electrons hitting the compound causing them to break off into fragments. [14] The resulting data shows the signal areas of the impurity to give a raw profile of the compound. [8] In other words, the composition of the impurity can be determined using the GC-MS method. Therefore, the simultaneous identification of a group of synthetic markers will determine the synthetic pathway used in the manufacturing of the sample. For example, if the following impurities were present: N-cyclohexyl-acetamide, N,N-Dimethylpiperonylamine, 3,4-Methylenedioxyamphetamine, N-Methylpiperonylamine, 2-Methyl-(6,7-methylenedioxyphenyl)-3-methylmorpholine, 3-Methyl-6,7-methylenedioxyisoquinoline-1,4-dione, the sample was synthesized from MDP-2-P by reduction of 1-(3,4-methylenedioxyphenyl)-2-nitropropene from piperonal, and the MDMA was synthesized by reductive amination. [9] Since N-cyclohexylacetamide was the product of a catalyst and acetic acid, the compound can be any N-alkylacetamide corresponding to the amine used as the catalyst; thus the synthetic pathway would still be reductive amination of MDP-2-P from piperonal. [9]Palhol et al. analyzed 29 extracted impurities from 52 seized tablets using methylene chloride under basic conditions. [15] The extracts were then analyzed by GC with flame ionization detection to identify the sample’s precursors, intermediates, and by-products. [15] The components separated from the GC pass through a hydrogen flame to ionize the gas in order to determine its concentration. [16] The results of GC-FID were confirmed through GC-MS. The precursor was found to be MDP-2-P as it was found in over three fourths of the tablets. The intermediates and by-products suggested that most of the MDMA was synthesized via reductive amination, while the rest were done through Leukart synthesis. The similar impurities suggested that the tablets were manufactured by the same laboratory. It was found to be similar to seized batches from Northern France in previous years. [15]
Kochana et al. determined that the use of Solid Phase Extraction-Thin Layer Chromatography (SPE-TLC) gave the best results when profiling 1-(3,4-methylenedioxyphenyl)-2-nitropropene from piperonal. [17] This compound can be reduced to give MDP-2-P, the precursor for MDMA in both Leukart and reductive animation synthesis. The impurity was extracted in C18 columns, and TLC separation occurred on silica gel 60 plates with fluorescent indicator F254. It was concluded that a concentrated extraction provided better profiling, and that a 2:8 acetonitrile-chloroform mixture was the best possible mobile phase for TLC separation. [17] However, the limitations of this analytical technique reduce the possibility of the its use in the real world as it only applies to the 1-(3,4-methylenedioxyphenyl)-2-nitropropene intermediate.
Carter et al. had used GC-Isotope Ratio Mass Spectrometry to analyze the δ13C values for extracted MDMA from four tablets. [18] The MDMA was then oxidized to MDP-2-P and piperonal of which the δ13C values were taken. Since the δ13C value was more concentrated for piperonal than MDP-2-P, it was suggested that the MDMA tablets were synthesized from piperonal and not from the oxidation of isosafrole. [18] A follow up study by Carter et al. determined the δ13C, δ2H, and δ15N values of MDMA from five different seized tablets. [19] Based on the results, when considering the δ13C values, three batches were isotopically distinct. Four batches were distinct based on δ2H. [19] However, each batch was completely distinct when all three values were taken into account. This suggests that the isotropic analysis of active ingredients in MDMA can be used as a “fingerprint” of the sample, which could be connected back to the original batch.
The four analytical techniques discussed above were GC-MS, GC-MS with flame ionization detection, SPE-TLC, and GC-ISRMS. Each of these techniques has their own advantages and disadvantages. The problem with GC-MS is that it does not work for non volatile components. However, it has been the most used method in impurity profiling so it does have a definitive procedure and protocol. Also, it provides an explicit analysis of what compounds are present in the sample. The use of flame ionization detection along with GC-MS allows for greater sensitivity towards hydrocarbons. [20] Flame ionization allows organic compounds with greater carbon atoms than nitrogen, phosphorus, or sulfur atoms to be detected better because of the sensitivity of FID to high carbon concentrations. [16] The disadvantage of FID would be that the sample would be destroyed. [16] However, in a situation where a large batch of MDMA would be seized, this would not be a problem. Moreover, the sensitivity of FID depends on certain aspects, such as the rate flow of the carrier gas, the rate flow of the combustion gas, the diameter of the jet the flame is produced by, and the position of the jet to the detector. [16] As such, if the compound is not a hydrocarbon, like a highly oxygenated compound, the sensitivity of FID decreases because the combustibility of the compound is lower. [20] The SPE-TLC technique was great when profiling a specific precursor, 1-(3,4-methylenedioxyphenyl)-2-nitropropene from piperonal, however, it would need to be modified for each impurity. So as an impurity profiling technique, it falls short in terms of practicality. GC-ISRMS provided the greatest amount of distinction between batches because it defined each batch according to three isotopic values, δ13C, δ2H, and δ15N. Although, it provided the best “fingerprint” of the MDMA samples seized, it was performed under the assumption that MDMA was oxidized from MDP-2-P and piperonal. After reviewing each of the techniques, GC-MS would be the best technique to identify the impurities associated with whichever synthetic pathways. Because the literature on GC-MS is already prevalent and it provides definitive results of the components in the sample, it would be a good identifier of impurities in a sample. From these impurities, the synthetic pathway can be deduced easier. But in order to identify if a sample was from a specific batch and the synthetic pathway has already been deduced, GC-IRMS would be best. Although superfluous, it would provide a strong support of the GC-MS results. Even though FID has the greatest sensitivity to identify impurities, it should be used as a last resort as this technique destroys the sample.
Cheng et al. used a combination of infrared spectroscopy, high performance liquid chromatography, liquid chromatrgraphy electrospray ionization mass spectrometry, and GC-MS to identify the impurities in 600,000 tablets seized in Hong Kong. [21] Their analysis found the most common impurities to be N-formyl MDMA, 3,4-methylenedioxyN-methylbenzylamine, 3,4-methylenedioxyphenyl-2-propanol, and MDP-2-P. From their results, reductive amination using cyanoborohydrate and the Leukart synthetic pathways were found to be most prevalent in the samples. [21] The use of multiple techniques to identify synthetic pathways of numerous samples seems to be most efficient because the limitations of one technique can be overcome through the use of another.
How to Identify the Presence of MDMA in an Individual
The identification of impurities of MDMA helped to determine which synthetic pathway was used to produce the tablet. But how would you identify if MDMA was in an individual’s system? Various analytical methods have been applied to samples of hair and urine to determine the presence of MDMA. Urine and hair samples are used mainly because they lack the complexities of blood or organ samples. [22] When comparing hair relative to urine samples, hair samples have a greater percentage of being positive to MDMA than urine. [23] In other words, MDMA will test positive in urine for frequent users, but will not test positive for occasional users. However, if hair is analyzed, occasional users would test positive for the compound. Opiates and amphetamines have a relatively short window of indication in urine, about two to three days; whereas hair can indicate upwards of three months, possibly longer. [24] The use of hair or urine sample depends on what is being analyzed. For instance, Lee et al. used hair samples to identify chronic use of MDMA in anorectics. [25] As such, the month by month use and amount of MDMA can be identified in hair samples since it grows at a rate of about one centimeter per month. [25] Although a urine sample would also determine the presence of MDMA in the system (if the anorectic was a frequent user), it would not provide the same information as a hair sample. A sample of urine or hair is tested for not only MDMA but their metabolites as well, such as 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-dihydroxymethamphetamine, 3-hydroxy-4-methoxymethamphetamine. [26] HMMA is the product of o-methylation of 3,4-dihydroxymethamphetamine and is considered the main metabolite of interest in urine. [26] However, since HMMA is easily hydrolyzed, a majority of the compound is excreted as glucuronide or sulfate so it is difficult to analyze for HMMA. [27]The analytical technique usually associated with urine and hair analysis is GC-MS. [22] In order for the samples of hair and urine to be analyzed, they must be prepared for GC-MS. [23] The method utilized by Han et al. on the urine samples was to use fluorescence polarization immunoassays followed by GC-MS analysis. [23] The hair was first washed twice with water than twice by methanol, incubated in methanol with 1% hydrochloric acid, evaporated, incubated with ethyl acetate and TFAA, cooled, evaporated, and then mixed with methanol. [23] GC-MS was used to analyze the hair samples of 791 subjects that were suspected of using MDMA. [23] The results from GC-MS analysis identified 44 individuals with MDMA or MDA in their hair, and from that, 9 were identified with MDMA or MDA in their urine. [23] Although a successful procedure, high performance liquid chromatography with a fluorescence detector could be used in place of GC-MS when analyzing urine. [28] MDMA, along with many other methylenedioxylated amphetamines, fluoresces. [22] As such, a fluorescence detector would have high selectivity towards such fluorescent compounds. Since these compounds naturally fluoresce, the derivation process in GC-MS would not be required. [28] This would decrease the cost of analyzing urine, making it more accessible to companies and countries with limited funds. [22] Another cost saving method would be gas chromatography coupled with flame ionization detection. [29] Although not as sensitive as GC-MS, GC-FID is sensitive enough to detect MDMA, MDA, and MA in urine samples. [29] It reduces the cost of identifying the compound as most laboratories have the equipment to carry out the procedure. Furthermore, by coupling GC-FID with solid phase extraction, the concentration of MDMA, MDA, and MA can be determined from urine samples. [30] Based on the components found in the samples, the purity of the tablets can also be determined. [29] For instance, if only MDMA was found after SPE and GC-FID, the tablet consumed was purely MDMA if time of collection of the sample was taken into account. [30] In other words, if the sample collected took some time to be analyzed, some of the other compounds, like MDA, could have been reduced to levels undetectable by GC-FID. For hair analysis specifically, liquid chromatography-mass spectrometry followed by mass spectrometry is becoming more popular than GC-MS because of its increased sensitivity along with lack of product loss during the derivatization step. [25] Due to the volatility associated with amphetamines, during the derivatizaiton step, evaporation occurs, leading to the loss of MDMA and inaccurate results. [25]
Conclusion
MDMA, a classified illegal enactogen, has become a prevalent recreational drug as it induces increased serotonin release while hindering synaptic reuptake. [5] The surge in popularity of the drug is accompanied by its production in clandestine laboratories. As such, methods to determine the synthesis of MDMA by analyzing the impurities associated with a sample has linked certain batches of seized MDMA to certain labs [8-11]. Various methods of analyzing the impurities include GC-MS, GC-MS with FID, SPE-TLC, and GC-IRMS. By considering a group of impurities together, the synthetic pathway can be determined [9]. Although much has been accomplished MDMA analysis, further research is needed to determine more impurities associated with various creative synthetic pathways. Not only can MDMA be analyzed in impurity profiling, but the presence of MDMA can be analyzed in human urine and hair. While GC-MS is the prevalent method, several other techniques have become increasingly popular such as GC-FID and LC-MS/MS as new approaches are being explored. [25]References
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